scholarly journals A Self-Priming Microfluidic Chip with Cushion Chambers for Easy Digital PCR

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 158
Author(s):  
Gangwei Xu ◽  
Huaqing Si ◽  
Fengxiang Jing ◽  
Peng Sun ◽  
Dongping Wu
Keyword(s):  
Microfluidic Chip ◽  
Low Cost ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Pdms Layer ◽  
Resource Limited ◽  
Dna Template ◽  
Digital Polymerase Chain Reaction ◽  
Easy Operation

A polydimethylsiloxane (PDMS)-based self-priming microfluidic chip with cushion chambers is presented in this study for robust and easy-operation digital polymerase chain reaction (dPCR). The chip has only one inlet and can partition samples autonomously through negative pressure, provided by a de-gassed PDMS layer with a multi-level vertical branching microchannel design. Meanwhile, cushion chambers make the chip capable of very robust use for sample partitioning. Finally, the proposed microfluidic chip showed excellent performance in the absolute quantification of a target gene by performing quantitative detection of a 10-fold serial dilution DNA template. Owing to its characteristics of easy operation, low cost, and high robustness, the proposed dPCR chip is expected to further promote the extensive application of digital PCR, especially in resource-limited settings.

scholarly journals A Double-Deck Self-Digitization Microfluidic Chip for Digital PCR

Micromachines ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1025
Author(s):  
Gangwei Xu ◽  
Huaqing Si ◽  
Fengxiang Jing ◽  
Peng Sun ◽  
Dan Zhao ◽  
...  
Keyword(s):  
Microfluidic Chip ◽  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Mutual Interference ◽  
Fluorescence Signal ◽  
Pdms Layer ◽  
Glass Substrates ◽  
Planar Area ◽  
Dna Template

In this work, a double-deck microfluidic chip was presented for digital PCR application. This chip consists of two reverse-placed micro-patterned polydimethylsiloxane (PDMS) layers between the top and bottom glass substrates. Each micropatterned PDMS layer contains more than 20,000 cylindrical micro-chambers to hold the partitioned droplets. The double-deck designs can double the number of chambers and reagent capacity without changing the planar area of the chip. In addition, carbon black was mixed into the pure PDMS gel to obstruct the passage of fluorescence from the positive chambers between the two layers of the chip. Thus, the fluorescence signal of micro-chambers in different layers of the chip after PCR can be collected without mutual interference. The quantitative capability of the proposed chip was evaluated by measuring a 10-fold serial dilution of the DNA template. A high accuracy of the absolute quantification for nucleic acid with a dynamic range of 105 was demonstrated by this chip in this work. Owing to its characteristics of small planar area, large capacity, and sensitivity, the double-deck microfluidic chip is expected to further promote the extensive applications of digital PCR.

scholarly journals Clarity Plus™ digital PCR: A novel platform for absolute quantification of SARS-CoV-2

2021 ◽  
Author(s):  
Shawn Yi Han Tan ◽  
Milton Sheng Yi Kwek ◽  
Huiyu Low ◽  
Yan Ling Joy Pang
Keyword(s):  
Dynamic Range ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Viral Loads ◽  
Nucleic Acid Analysis ◽  
Polymerase Chain ◽  
The Absolute ◽  
Assay Precision ◽  
Digital Polymerase Chain Reaction ◽  
Higher Sensitivity

In recent years, the usage of digital polymerase chain reaction (dPCR) for various clinical applications has increased exponentially. Considering the growing demand for improved dPCR technology, the Clarity Plus™ dPCR system which features enhanced multiplexing capability and a wider dynamic range for nucleic acid analysis was recently launched. In this study, a dPCR assay optimized for use on Clarity Plus™ was evaluated for the absolute quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent responsible for the global coronavirus disease 2019 (COVID-19) outbreak. The assay demonstrated good inter- and intra- assay precision, accuracy, as well as excellent linearity across a range of over 6 orders of magnitude for target gene quantification. In addition, comparison of the assay on both dPCR and qPCR platforms revealed that dPCR exhibited a slightly higher sensitivity compared to its qPCR counterpart when quantifying SARS-CoV-2 at a lower concentration. Overall, the results showed that the dPCR assay is a reliable and effective approach for the absolute quantification of SARS-CoV-2 and can potentially be adopted as a molecular tool in applications such as detecting low viral loads in patients as well as in wastewater surveillance of COVID-19.

A microfluidic chip based on surfactant-doped polydimethylsiloxane (PDMS) in a sandwich configuration for low-cost and robust digital PCR

2017 ◽  
Vol 245 ◽  
pp. 414-422 ◽  
Author(s):  
Yayun Fu ◽  
Hongbo Zhou ◽  
Chunping Jia ◽  
Fengxiang Jing ◽  
Qinghui Jin ◽  
...  
Keyword(s):  
Microfluidic Chip ◽  
Low Cost ◽  
Digital Pcr ◽  
Sandwich Configuration

scholarly journals Development of a Droplet Digital Polymerase Chain Reaction for Sensitive Detection of Pneumocystis jirovecii in Respiratory Tract Specimens

2021 ◽  
Vol 8 ◽  
Author(s):  
Jie Yi ◽  
Nan Wang ◽  
Jie Wu ◽  
Yueming Tang ◽  
Jingjia Zhang ◽  
...  
Keyword(s):  
Respiratory Tract ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Pneumocystis Pneumonia ◽  
Pneumocystis Jirovecii ◽  
Immunocompromised Patients ◽  
Balf Sample ◽  
Bronchoalveolar Fluid ◽  
Life Threatening ◽  
Digital Polymerase Chain Reaction

Background:Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR.Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR.Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR.Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii. Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.

scholarly journals Application of digital PCR (dPCR) in the detection of Covid-19 in food

2021 ◽  
Vol 271 ◽  
pp. 02022
Author(s):  
Hua Liu ◽  
Shanti dwita Lestari
Keyword(s):  
Pathogenic Bacteria ◽  
Low Cost ◽  
Virus Detection ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Detection Methods ◽  
Multiple Target ◽  
Detection Technology ◽  
Virus Safety ◽  
Pcr Technology

Covid-19 detection in food is an effective solution to ensure the accurate detection rate of Covid-19. The difficulties and detection methods of food virus safety detection and the feasibility of digital PCR detection technology are analyzed. The main parameters and characteristics of dPCR technology and other PCR technologies are compared. The application of dPCR technology in the detection of food viruses and pathogenic bacteria, the application of dPCR technology in the preparation and purity verification of Covid-19 RNA reference material, and the steps and methods of dPCR technology in food testing Covid-19 were expounded. Compared with traditional detection methods, digital PCR technology has great advantages in virus detection limit and stability. dPCR will develop towards high flux and automation, and achieve the absolute quantification of multiple target sequences at low cost. It will help to play a crucial role in the detection of covid-19 in food.

A rapid nucleic acid concentration measurement system with large-field-of-view for droplet digital PCR microfluidic chip

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Jinrong Shen ◽  
Jihong Zheng ◽  
Zhenqing Li ◽  
Yourong Liu ◽  
Fengxiang Jing ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr ◽  
Field Of View ◽  
Large Field ◽  
Chain Reaction ◽  
Effective Technique ◽  
Nucleic Acid Concentration ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Droplet digital polymerase chain reaction(ddPCR) is an effective technique for the absolute quantification of target mucleic acid unparalleled sensitivity. However, current commerical ddPCR device for the detection of the gene...

scholarly journals Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Ian D. Woolsey ◽  
Berit Blomstrand ◽  
Øivind Øines ◽  
Heidi L. Enemark
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Dna Content ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Seeding Density ◽  
Modern Methods ◽  
Freely Suspended ◽  
Dna Template ◽  
Template Dna

Abstract Background Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA. Methods We assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106–1); (ii) diluted template DNA from pure oocysts (×10–×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105–1 and 5 × 104–50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10–×1000). Results Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations. Conclusions For many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

scholarly journals Pre-Degassed Microfluidic Chamber-Based Digital PCR Device for Meat Authentication Applications

Micromachines ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 694
Author(s):  
Hezhi Hu ◽  
Jingmeng Cheng ◽  
Chunyang Wei ◽  
Shanshan Li ◽  
Chengzhuang Yu ◽  
...  
Keyword(s):  
Microfluidic Device ◽  
Dna Amplification ◽  
Digital Pcr ◽  
Copy Numbers ◽  
Microfluidic Chamber ◽  
Skilled Personnel ◽  
Amplification Process ◽  
Dna Template ◽  
Pilot Channel ◽  
Digital Polymerase Chain Reaction

Droplet digital polymerase chain reaction (ddPCR) suffers from the need for specific equipment and skilled personnel; thus, we here present a chamber-based digital PCR microfluidic device that is compatible with fluorescence image read-out systems and removes bubbles by a pre-degassed microfluidic device that consists of a pilot channel and micro chamber arrays. Digitalized PCR reagents are introduced into micro chambers, and thermocycles are taken to perform a DNA amplification process. Then, fluorescence images of a micro chamber array are read out and analyzed to obtain the total number of positive chambers. Thereby, the copy numbers of target DNA are calculated for quantitative detections. As a validation, this device is evaluated by the application of meat authentication. We performed dPCR tests using DNA templates extracted from a pure mutton DNA template with different dilutions. Then, the dPCR chip was used to identify the meat authentication using mutton–chicken mixtures with different mass ratios, showing its performance in real biotechnical applications.

Absolute quantification of DNA methylation using microfluidic chip-based digital PCR

2017 ◽  
Vol 96 ◽  
pp. 339-344 ◽  
Author(s):  
Zhenhua Wu ◽  
Yanan Bai ◽  
Zule Cheng ◽  
Fangming Liu ◽  
Ping Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Microfluidic Chip ◽  
Digital Pcr ◽  
Absolute Quantification

Simple structured polydimethylsiloxane microvalve actuated by external air pressure

2006 ◽  
Vol 220 (8) ◽  
pp. 1283-1288 ◽  
Author(s):  
S-W Lee ◽  
D-J Kim ◽  
Y Ahn ◽  
Y G Chai
Keyword(s):  
Fluid Flow ◽  
Microfluidic Chip ◽  
Glass Substrate ◽  
Low Cost ◽  
Injection Pressure ◽  
Air Pressure ◽  
Negative Photoresist ◽  
Plasma Surface ◽  
Plasma Surface Treatment ◽  
Easy Operation

This article describes a simple structured microvalve made of polydimethylsiloxane (PDMS) and glass. For easy operation, the microvalve is actuated by external air pressure. The microvalve is fabricated by replica moulding using a negative photoresist (SU-8) as the mould. For low cost and biocompatibility, PDMS is used as the chip material. The PDMS valve is bonded to a Pyrex glass substrate using an O2 plasma surface treatment. It was found that no leakage occurs under a valve-actuating air pressure of 0.35 MPa when the injection pressure of liquid in the microchannel of the valve is 0.01-0.04 MPa. The fabricated microvalve is applied to a microfluidic chip having one inlet and two outlet channels. Fluid flow is controlled by the microvalve.

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